The broad long-term objective of this project is to understand the couple interactions among the smooth muscle thin filament protein components, actin, tropomyosin, caldesmon and myosin that are involved in the cooperative activation and relaxation processes of smooth muscle contraction. Specifically, we aim to: 1) understand the difference between the activating properties of smooth muscle compared to striated muscle tropomyosin in terms of the end-to-end interactions between tropomyosins along the thin filament; 2) determine the steps in the actomyosin ATPase cycle that are affected by phosphorylation; 3) explain the "latch" state of smooth muscle, whereby the muscle can maintain force over long times without much ATP usage at low degrees of phosphorylation; 4) Determine if caldesmon functions in smooth muscle in an analogous manner to troponin in striated muscle and explore the role of other components in the regulation process. The methods to be used include ATPase activity measurements, kinetics and equilibrium titrations with fluorescence probes on the protein components. Tropomyosin isoforms isolated from smooth muscle and mutated tropomyosins will be used. With the information obtained from these studies the basic mechanisms of smooth muscle regulation will be better understood so that the several smooth muscle abnormalities can be characterized.